Elsevier

Vaccine

Volume 17, Issue 1, January 1999, Pages 95-98
Vaccine

A model to study the effects of a viral inactivator (β-propiolactone) on DNA ligation and gene expression in E. coli and Cos cells

https://doi.org/10.1016/S0264-410X(98)00087-5Get rights and content

Abstract

An experimental model to study the effects of viral inactivators on the biological properties of DNA was developed. β-propiolactone (βPL) was used in this model and its effects on ligation, transfer and gene expression of naked DNA were assessed. Evidence that βPL impairs these two major DNA functions are presented. The amounts of βPL that alter or abolish gene expression and prevent DNA cohesive ends ligation were determined. Based on these observations, it was concluded that this experimental approach could be used to study the effects on the biological properties of DNA of other inactivators used in vaccine preparations.

Introduction

The use of mammalian continuous cell lines to produce biologicals for human use has raised the issue that the residual cellular genomic DNA contained in the final product might be of potential oncogenic risk1, 2. This issue has precluded the use of transformed cell lines to produce vaccines for humans.

The effects of viral inactivators on DNA properties of ligation and gene expression through which a foreign DNA might randomly integrate into the host cell DNA and eventually leads to oncogenicity, have not been investigated. We have developed an experimental model where two expression systems, a prokaryotic and a eukaryotic one, were used to study the effect of β-propiolactone (βPL) treatment on gene expression and DNA ligation. Our model uses a closed circular and/or linear form of an expression vector with a known reporter gene. This DNA is treated with a viral inactivator, religated (the linear form) and assayed for the expression of the reporter gene in the respective expression system. The alkylating agent βPL was chosen as viral inactivator because it is commonly used in vaccine preparations[3]. It is also the most studied among the chemicals used as viral inactivators. Several reports have shown that βPL reacts irreversibly with a variety of macromolecules such as proteins and nucleic acids4, 5. It has previously been shown that βPL treatment impairs irreversibly the DNA properties of hybridization and duplication[6].

Section snippets

Bacterial strain E. coli JM109

rec A1, End A1, gyr A96, thi hsd R17, Sup E44, D(LacproAB), F tra D36, proAB, laciq, ZDM15 was used to host the pGEM7 plasmid (Promega-France) expressing the β lactamase gene (ampicillin resistance gene).

βPL treatment

Plasmids DNA (pGEM7 and CDM8/CD8α) resuspended in 10 mMTris/1 mM EDTA, pH 7.5 (TE), was treated by various concentrations (0.01%, 0.025%, 0.05% and 0.1% V/V) of βPL for 2 h at room temperature, 16 h at 4°C and a 2 h incubation at 37°C to complete the inactivation of βPL. This procedure is used for

Results

To investigate the effect of βPL on gene expression in procaryotic cells, the pGEM7 vector carrying the β lactamase encoding gene was treated with βPL concentrations ranging from 0.01% to 0.1% (V/V) and introduced into competent (106 clones per μg of DNA) E. coli JM109 cells. Clones expressing the β lactamase gene were able to grow on LB agar plate supplemented with ampicillin. A negative correlation was observed between the number of clones expressing the ampicillin resistance gene and

Discussion

While βPL action on non-naked DNA structure is well documented4, 5, 11, only assumptions could be made on its action on DNA ligation and effective gene transfer and expression. In this report, we show that βPL impairs gene expression and ligation of naked DNA. Our data argue in favor of an alteration of gene expression and ligation as a consequence of βPL treatment. The extent of this alteration is proportional to the amount of βPL interacting with the DNA. βPL treatment could prevent the DNA

Acknowledgements

We are grateful to Mr B. Kaabi for his help with the statistical analysis.

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